Hybridoma cell strain capable of secreting bisamides resistant monoclonal antibody and application thereof

[0060] Example 1: Synthesis of Halfactic (Hapten 1)

[0061] Since the small molecules do not have immunogenicity, it is not possible to stimulate the mice to produce an immune response, and then the antibody is generated, and the small molecule is counted to the protein by protein connection technology, which is commonly used in protein coupling techniques. The active group has an amino group, a carboxyl group, a hydroxyl group, a mercapto group. In order to obtain a monoclonal antibody that can specifically recognize cyclomide, bromodoacetamide, chlorotalamide, cecutamide, therefore needs to be designed and derived A good semi-antigen, the specific derivative steps are as follows.

[0062] Step 1: 4- (2-amino-3-bromo-5-chloroflanionlamino) butyl tert-butyl tert-butyl

[0063]

[0064] 1.0 g (4.0 mmol) 2-amino-3-bromo-5-chlorobenzoic acid, 10 ml of toluene and 0.80 ml (12.0 mmol) thionyl chloride were added to the reaction flask, and the solvent was removed, and the solvent was removed. 5 ml Using the above-mentioned spare solution into a reaction bottle containing 10 ml of tetrahydrofuran, 1.7 mL (12.0 mmol) triethylamine and 2.3 g (12.0 mmol) 4-aminobutyric acid tert-butyl, at room temperature for 0.5 hours, TLC showed the reaction completion . After the solvent was removed, 50 ml of water and 200 ml of ethyl acetate were added to isolate extraction. The organic phase was washed with 100ml saturated brine, with anhydrous NA 2 SO 4 Dry, remove under reduced pressure. The residue was purified from the column chromatography (4- (2-amino-3-bromo-5-chlorofinoen) butyrate tert-butyl oxyhyric acid) 1.4 g.

[0065] Step 2: Synthesis of Haldactor Hapten 1

[0066]

[0067] 1.0 g (3.3 mmol) of 3-bromo-1- (3-neopropyridin-2-yl) -1H-pyrazole-5-carboxylic acid, 10 ml toluene and 0.68 ml (10 mmol) of thionyl chloride reactive flask Removal of 3 hours, remove the solvent, and add 4 ml of hydrogenfuran; the above-mentioned spare solution is dropped into 10 mL of tetrahydrofuran, 1.39 mL (10 mmol) Triethylamine and 480 mg (1.1 mmol) 4- (2-amino-5-chlorobenzoyl amino) In the reaction bottle of tert-butyl butyl butyrate, the mixture was stirred at room temperature for 0.5 hours, and the TLC showed the reaction. After removing the solvent, 50 ml of water and 200 ml of ethyl acetate were added to isolate extraction; the organic phase was washed with 100 ml of saturated brine, with anhydrous NA 2 SO 4 Dry, remove under reduced pressure. The residue was purified by column chromatography to obtain a crude product, dissolved in 5 mlTFA and DCM mixed solution (V / V = ​​1: 1), and stirred at room temperature for 0.5 h. The solvent was removed under reduced pressure, and the residue was purified from the column chromatography to give a 190 mg product. The characterization result is: 1 H NMR (400 MHz, Methanol-D4) Δ8.44 (DD, J = 4.7, 1.6 Hz, 1H), 8.03 (DD, J = 8.1, 1.6 Hz, 1H), 7.75 (D, J = 2.3 Hz, 1H) 7.52 (DD, J = 8.1, 4.7 Hz, 1H), 7.48 (D, J = 2.3 Hz, 1H), 7.32 (S, 1H), 3.28-3.24 (M, 2H), 2.29 (T, J = 7.3 Hz, 2H), 1.84-1.71 (m, 2H)., ESI-MS: C 20 Hide 15 BR 2 CL 2 N 5 O 4 + NA + Required 639.8766, Found 639.76. (Nuclear Magnetic Schematic Figure 2 - Figure 3 )

[0068] Example 2: Complete antigen synthesis

[0069] 2.8 mg of semi-antigen (Hapten 1), 1.6 mg N-hydroxy succinimide (NHS), dissolved in 300 μl N, N-dimethylformamide (DMF), at room temperature for 10 min; again, 2.6 mg 1- (3-dimethylaminopropyl) -3-ethyl carbon diimide hydrochloride (EDC), after 100 μl of DMF, adding to a semi-anti-stock solution, stirring at room temperature for 6-8 h (called A liquid). 10 mg of BSA was diluted with 0.01 M carbonate buffer (CBS) to 5 mg / ml (referred to as a B liquid), and then slowly added A. The solution was slowly added to the B liquid, and the room temperature was reacted overnight; then 0.01M PBS solution Dialysis, the unreacted small molecular half antigen was removed, and the complete antigen was obtained, and the identification was performed by an ultraviolet absorption scanning method.

[0070] Example 3: Synthesis of the package

[0071]To improve sensitivity, is to design a new packet (Hapten2) hapten with the following structure as shown:

[0072]

[0073] Hapten (Hapten2) method analogous to the synthesis of hapten (Hapten1). Synthesis of specific original coating steps to: 3.6mg hapten (Hapten2), 2.3mgN- hydroxysuccinimide (NHS) was dissolved in 300μL of anhydrous N, N- dimethylformamide (DMF), the reaction was stirred at room temperature 10min, to give hapten solution; 3.8mg 1- (3- dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was dissolved in anhydrous 100μL of DMF, the solution to the hapten , 6-8h the reaction was stirred at room temperature to give a solution a; and 10mg chicken ovalbumin (OVA) with 1mL at a concentration of 0.01mol / L carbonate buffer (CBS) was diluted to give a solution B; solution a dropwise to solution B was slowly added to the reaction to obtain a reaction solution; the reaction solution was dialyzed with PBS solution to remove unreacted small hapten molecules, is obtained the original packet. Wherein the hapten (Hapten2) Characterization data: 1H NMR (400MHz, DMSO-d6) δ12.52 (s, 1H), 12.13 (s, 1H), 9.02 (s, 1H), 8.54 (dd, J = 4.7 , 1.6Hz, 1H), 8.30-8.15 (m, 2H), 7.93 (d, J = 2.4Hz, 1H), 7.67 (dd, J = 8.1,4.7Hz, 1H), 7.55 (dd, J = 8.9, 2.4Hz, 1H), 7.19 (s, 1H), 3.33 (q, J = 6.0Hz, 2H), 2.32 (t, J = 7.3Hz, 2H), 1.79 (p, J = 7.2Hz, 2H) .ESI -MS: C 20 Hide 16 BrCl 2 N 5 O 4 + H + required 539.9841, found 539.89. (see NMR characterization Figure 4 - Figure 5.